Our aim is to study the organization of neurotransmitter receptors on nerve and muscle cells in relationship to the development and function of synapses. We have used Alpha-bungarotoxin as a specific probe for the visualization and quantitation of nicotinic acetylcholine receptor sites. Our recent work has focused upon the factors, extrinsic and intrinsic to the developing skeletal muscle fiber, which regulate the distribution of nicotinic acetylcholine receptors. We have now used image intensification to directly observe the early steps (30 minutes to 2 hours) of acetylcholine receptor aggregation, induced by neuronal factors, on muscle fibers grown in culture. The process is characterized by the initial formation of microaggregate clusters which later become dense receptor aggregates. Electron microscopy of directly observed myotubes indicates that specializations of the extracellular matrix (basal lamina), and of the cytoskeleton (a microfilament network and a dense submembrane bar) are enriched in receptor aggregate regions within 2 hours after the aggregates form and that the specialized cell surface often shows deep invagination.